This invention relates to a novel deoxyribonuclease capable of cleaving DNA independently from divalent cations under acidic conditions, a DNA encoding same and use of these for the prophylaxis and treatment of infectious diseases as well as for the treatment of cystic fibrosis.
The presence of various deoxyribonucleases (hereinafter referred to as DNase) in mammalian cells has been known. DNase II is one of the DNases studied most and catalyzes DNA hydrolysis reaction in the absence of divalent cations at acidic pH [in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 4, pp. 271-287 (1971), Academic Press, New York; Arch. Biochem. Biophys., 300: 440-450 (1993)]. While the acid DNase activities are widely found in various animal tissues [Biochim. Biophys. Acta, 1119: 185-193 (1992); J. Biol. Chem., 273: 2610-2616 (1998)], DNase II has been considered to be the sole enzyme responsible for the acid DNase activities. Because DNase II shows low organ specificity and is distributed ubiquitously, a possibility of DNase II playing an important biological role in the fundamental biological phenomena, such as DNA catabolism and apoptosis, has been suggested [The Enzymes (1971), supra; Arch. Biochem. Biophys., 300: 440-450 (1993)].
Even though the enzymological properties of the DNase II isolated from different organisms are very similar, their physicochemical properties and molecular structures are strikingly different. For example, it is known that porcine DNase II is a complex protein consisting of unidentical subunits derived from its precursor protein, but DNase II derived from other animals are mostly single polypeptides [J. Biol. Chem., 260: 10708-10713 (1985); Biochem. Biophys. Res. Commun., 247: 864-869 (1998); J. Biol. Chem., 251: 116-123 (1976); Gene, 215: 281-289 (1998)]. Furthermore, the apparent molecular weights of DNase II vary from 26.5 kDa to 45 kDa [J. Biol. Chem. (1976), supra; Gene, (1998), supra; J. Biol. Chem., 247: 1424-1432 (1972); Eur. J. Biochem., 202: 479-484 (1991)].
The diversity of acid DNases can be also appreciated from the subcellular localization. DNase II is considered to be localized in lysosomes [J. Biol. Chem. (1972), supra; Biochim. Biophys. Acta, 1007: 15-22 (1989)], but acid DNase activity is also found in nuclear fraction [Arch. Biochem. Biophys. (1993), supra; Biochem. J., 136: 83-87 (1973)].
The reason for such molecular diversity of DNase II still remains unclear, but the aforementioned findings suggest the existence of a different acid DNase distinguishable from DNase II. In fact, the present inventors have identified and partially purified novel acid DNases (DNase xcex1 and DNase xcex2) from the nuclear fraction of rat thymus (JP 8-187079 A). In view of the foregoing situation it is considered to be critical for the elucidation of the diversity of acid DNases to search other novel acid DNases and determine their characteristics.
In addition, DNase has been actively studied with the aim of applying same for the prophylaxis and treatment of various diseases. One of the clinical applications of DNase, which has been drawing particular attention in recent years, is an application to the treatment of cystic fibrosis (hereinafter sometimes to be also referred to as CF) [Annu. Rev. Pharmacol. Toxicol., 35: 257-276 (1995); Chest, 107: 65-70 (1995)]. CF is a lethal hereditary disease caused by abnormal chloride ion channel of exocrine glands. In the Caucasian population, one in 2500 newborns suffers from this disease and one in 25 Caucasians is a carrier. About 90% of the CF patients die of respiratory insufficiency caused by intractable infection with Pseudomonas aeruginosa in the inferior airway in their 20""s and 30""s [Curr. Opin. Pulm. Med., 6: 425-434 (1995)]. Phlegm that is accumulated in the airway to impair the respiratory function is caused by high concentration DNA released from the disrupted leukocytes infiltrating into the inflammatony site. Genentech, Inc. U.S. is selling a recombinant DNase I as a therapeutic agent for CF in Europe and America, which aims at removing the high molecular weight DNA accumulated in the lung, recovering the respiratory function and preventing infectious diseases [Annu. Rev. Pharmacol. Toxicol. (1995), supra; Chest (1995), supra]. DNase I not only degrades DNA, but also depolymerizes F-actin which is abundant in the sputum of CF patients. However, since the resulting monomeric G-actin strongly inhibits DNase I, DNase I is immediately inactivated. Actually, DNase I hardly shows any therapeutic effect. Some attempts have been made to produce a G-actin nonsensitive DNase I by genetic recombination, but satisfactory DNase has not been obtained yet [Proc. Natl. Acad. Sci. USA, 93: 8225-8229 (1996); J. Biol. Chem., 273: 18374-18381 (1998)]. Thus, there is a demand on the identification of a novel G-actin nonsensitive DNase effective for the treatment of CF.
A second interest in the clinical application of DNase is that for the prophylaxis and treatment of infectious diseases. Some DNases are considered to play an important role in the biological defense mechanisms against infection with bacteria and viruses, based on degradation of foreign genomic DNAs. Accordingly, identification of the DNase involved in the prevention of infection in mammals, such as human, and utilization thereof as a medicament are expected to open a new possibility in the prophylaxis and treatment of infectious diseases.
It is therefore an object of the present invention to provide a novel acid DNase and clarify the characteristics of the enzyme, thereby providing critical information for the study of the molecular diversity of acid DNases. It is another object of the present invention to provide a novel G-actin nonsensitive DNase that can be effectively used as a therapeutic agent of CF. It is yet another object of the present invention to provide a novel DNase useful for the prophylaxis and treatment of infectious diseases.
In an attempt to accomplish the above-mentioned objects, the present inventors have conducted intensive studies, and succeeded in isolating cDNA clones containing an ORF encoding a novel protein homologous to human DNase II, from RNA derived from the liver of human, mouse or rat. Furthermore, it has been confirmed that this protein has an endonuclease activity capable of cleaving the DNA independently from divalent cations under acidic conditions, like DNase II, but is a novel acid DNase distinguishable from DNase II in the capability of exerting the DNase activity even in the neutral pH range and the sensitivity against divalent metallic ion inhibitors, as a result of the analysis of the physicochemical and enzymological characteristics of the protein obtained by culturing a host cell transformed with an expression vector containing the cDNA clone and purifying the recombinant protein. Then, the present inventors have designated the novel acid DNase as DLAD (DNase II-Like Acid DNase). The present inventors have also demonstrated that this enzyme has a high possibility of making an effective therapeutic agent of CF by confirming that the DLAD activity is not inhibited by G-actin. Moreover, the present inventors have confirmed a high possibility of the DLAD having a preventive effect on viral infectious diseases, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) A DNase which is an endonuclease capable of cleaving DNA independently from divalent cations under acidic conditions and having the following properties:
(1) active pH range: ca. 4.0 to ca. 7.6
(2) DNA cleavage mode: 3xe2x80x2-P/5xe2x80x2-OH end forming type
(3) sensitivity against inhibitors:
(i) inhibited by Zn2+
(ii) not inhibited by G-actin
(2) The DNase of (1) above, further having the following properties:
(1) optimal pH: ca. 5.2
(2) molecular weight: ca. 55 kDa as a post-translational modification product (SDS-PAGE)
(3) localization: present in cytoplasm and extracellularly, rich in cytoplasm
(4) tissue specificity: specifically expressed in the liver.
(3) A DNase having the following polypeptide (a) or (b):
(a) a polypeptide consisting of an amino acid sequence of amino acid Nos. 1 to 332 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1
(b) a polypeptide having the same amino acid sequence of (a) above, except that one to several amino acids are deleted, substituted, inserted, added or modified, wherein a mature protein has an endonuclease activity capable of cleaving a DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6.
(4) The DNase of any of (1) to (3) above, wherein a primary translation product contains an N terminal signal peptide sequence, preferably an amino acid sequence of the amino acid Nos. xe2x88x9222 to xe2x88x921 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 1.
(5) The DNase of any of (1) to (4) above, which is derived from a mammal, preferably mouse.
(6) A DNase having the following polypeptide (a) or (b):
(a) a polypeptide consisting of an amino acid sequence of the amino acid Nos. 1 to 334 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3.
(b) a polypeptide having the same amino acid sequence of (a) above, except that one to several amino acids are deleted, substituted, inserted, added or modified, wherein a mature protein has an endonuclease activity capable of cleaving a DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6.
(7) The DNase of any of (1), (2) and (6) above, wherein a primary translation product contains an N terminal signal sequence, preferably an amino acid sequence of the amino acid Nos. xe2x88x9227 to xe2x88x921 of the amino acid sequence shown in Sequence Listing, SEQ ID NO: 3.
(8) The DNase of (1), (2), (6) or (7) above, which is derived from a mammal, preferably human.
(9) A DNA encoding the DNase of any of (1) to (8) above.
(10) A DNA consisting of the following nucleotide sequence (a) or (b):
(a) a nucleotide sequence of the nucleotide Nos. 279 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2
(b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a DNase having an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6.
(11) A DNA consisting of the following nucleotide sequence (a) or (b):
(a) a nucleotide sequence of the nucleotide Nos. 213 to 1274 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 2
(b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a primary translation product of a DNase whose mature protein has an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6.
(12) The DNA of (10) or (11) above, which is derived from a mammal, preferably mouse.
(13) A DNA consisting of the following nucleotide sequence (a) or (b):
(a) a nucleotide sequence of the nucleotide Nos. 82 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4
(b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a DNase having an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6.
(14) A DNA consisting of the following nucleotide sequence (a) or (b):
(a) a nucleotide sequence of the nucleotide Nos. 1 to 1083 of the nucleotide sequence shown in Sequence Listing, SEQ ID NO: 4.
(b) a nucleotide sequence capable of being hybridized to the nucleotide sequence of (a) above under stringent conditions, which encodes a primary translation product of a DNase whose mature protein has an endonuclease activity capable of cleaving DNA independently from divalent cations in a pH range of from ca. 4.0 to ca. 7.6.
(15) The DNA of (13) or (14) above, which is derived from a mammal, preferably human.
(16) A recombinant vector containing the DNA of any of (9) to (15) above.
(17) An expression vector containing the DNA of any of (9) to (15) above and a promoter operably linked to said DNA.
(18) A transformant obtained by transforming a host cell with the expression vector of (17) above.
(19) A method for producing the DNase of any of (1) to (8) above, which comprises culturing the transformant of (18) above in a medium and recovering said DNase from the resulting culture.
(20) A pharmaceutical composition containing the DNase of any of (1) to (8) above, the expression vector of (17) above or the transformant of (18) above as an active ingredient.
(21) The pharmaceutical composition of (20) above, which is for the prophylaxis and treatment of infectious diseases or for the treatment of cystic fibrosis.
Inasmuch as the DLAD of the present invention is an acid DNase that expresses the activity in a broad pH range of from acidic to neutral pHs independently from divalent cations, and is resistant to G-actin, it is useful for degrading a high concentration DNA contained in the sputum of CF patients, improving the respiratory function.
Furthermore, because the DLAD of the present invention can suppress the intracellular expression of foreign genes, it also provides a useful means for the prophylaxis and treatment of infectious diseases, such as viral infection.